Project Summary: Di-iron carboxylate enzymes are utilized by nature to catalyze a wide variety of oxidation reactions, including the conversion of methane to methanol, the formation of desaturated fatty acids, and the generation of a tyrosyl radical that facilitates the biosynthesis of deoxyribonucleotides. Their divergent reactivities, however, belie their structural similarities. This proposal seeks to comprehend the metal-protein interactions that govern the reactivities of the di-iron carboxylate enzymes at a molecular level by developing a model system which accurately mimics the geometric, electronic, and catalytic properties of the natural enzymes. Advances in computational protein design have led to the development of a self-assembling four-helix bundle that contains a di-iron carboxylate active site (DFsc). This scaffold is well-suited for systematic investigations of the structure/function relationships found in the natural di-iron carboxylate enzymes as it is easily modifiable via point mutations and it mimics not only the first, but also the second and third, coordination spheres of the natural di-iron cluster. Structural characterization of DFsc, and variants will be undertaken utilizing X-ray crystallography. Subsequently, the redox potentials and catalytic abilities of these proteins will be determined and analyzed as a function of their structural variations. Finally, as a test of our understanding of the structure/function relationships present in the natural di-iron carboxylates, DFsc will be computationally redesigned to enhance its catalytic efficiency via stabilization of postulated catalytic intermediates. Relevance: Metalloenzymes are ubiquitous in biological systems and are required for a myriad of cellular processes. This research seeks to understand on a molecular level how nature harnesses the unique geometric and electronic properties of metal ions for catalytic activity. This knowledge can then be used to aid in the development of biomimetic catalysts. [unreadable] [unreadable] [unreadable]